Apparatus for propagation of culture germs



1927 F. J. MATCHETTE APPARATUS FOR YROPAGATION OF CULTURE GERMS 2 sheet s-sheet 1 Filed Jan, 1923 ME :JF? riemwrJ Mara/577's. W W

F. J. MATCHETTE APPARATUS FOR PROPAGATION OF CULTURE GERMS Feb. 22,1927. 1,618,461

Filed Jan. 2, 1923 2 Shoots-Sheet 2 EK4I :11

%IMW W Patente? Feb. 22, 1927.

UNITED STATES PATENT OFFICE.-

FRANK J. MATCEETTE, OI YORK, N. Y.

APPARATUS FOR iPBOPAG-ATION OF CULTURE GER'MS.

Application filed January teria. This bacteria is very rarelypresent in the soil and must therefore, be introduced by artificial inoculation.=: jFurther, these plants do not thrive well unles's'ninoculatd; and many crop failures .are d-irect1y. trac able to the absence of these bacterim The nitrogenous compounds formed re-i main in the soil with the rootsiof theplant after the crop .is harvested and materially contribute to the fertility of thefsoilfiin subsequent raising o'f other crops.- I :In. tact, nitrogen-fixing plants are often}. wnpri-' marily for the purpose of enriching't -for future use.

One process of artificial inoculation con-'- sists in diluting the bacteria carrying medium with'cle'anwater and moisteii in'g the seed with this solution just before planting. The bacteria then attach themselves to the roots of the.}. young"plant and r'n'ake'their permanent; home in the nod l 5 hi h they form on, theroots. -'-'f v 'The ordinary l': a.borat0rj ro cess agating these; nitrogen gathering which the germs-canlivje. This medium is put in test t'ubes,.'nursing flasks, or other suitable glas's.-'vessels.' These bacteria are aerobic in:;nature and 1 can only live and thrive while' in contact with air free of con-' taminating organisms. fiFor' this reason the food medium must bearranged to provide as large a surface as possible and this is usually accomplished by keeping medium is then inoculated with the socalled mother culture and the propagationl'movement begins.

It must period of preparation,inoculation and propagation, the containers andthe food medium must be maintained in an absolutely sterile condition at all times to avoid contamination of the culture by foreign organisms; It will be understood that every test tube or ba ter a consists; of providing f d on the con'-. The food that all airholes point in the same rotational be emphasized that during the 2, 1923. Serial No. 610,404.

other container must be handled in a like careful manner, and each one must be filled, sterilized, inoculated and tested frequently to determine the proper concentration of bacteria on a given area of food medium.

Apphcants invention is directed toward breeding .these legume bacteria on a commercial scale and to eliminate the cumbersome and tedious method, practical only for limlted laboratory production. Applicants apparatus also considerably shortens the breeding period for a given germ concentra- ,tion.' v

Infthe accompanying drawing: F1-gure;,1;shows the apparatus in elevation,.partlysectioned away to show interio "parts and their arrangement;

Fig. 2 isa plan view thereof; Fig. 3 isi a detail view of the air-distribut- 1ngsp1der;

ig. 4 is'a'diagram showing the arrangement-ot abat-tery of the apparatus;

Fig. 5 shows an'air-filter which is em- 'ployed during the process;

":Fig. 6 shows a filtering flask employed during the process of inoculation of the food medium with the mother culture.

1 and 2, the apparatus consists of tank 10 having a cover 11. This cover is provided Referring now more particularly to Figs.

with a ring 12 forming an opening in the center large enough for convenient cleaning -and also for the insertion and removal of the air distributing spider .18. On this ring is a suitable ridge 13 which forms an airtight packing when the cover 13 is screwed down nto position by means of the studs and nuts 14.. I

A" pipe 16 is carried by the cover 13 This pipe slidably fits into a suitable socket in the spider body 17 carrying'suitable pipes 18 which may be arranged in a variety of shapes, small air openings 19 being rovided Ion the sides of the pipes. It will e noted direction; thiswillcause a slight rotational of the entire body of liquid in the tank, and thus a more uniform aeration.

A suitable valve 20 is providedfo'r regulating the entrance of compressed air. compressed air passes through filter 21, "which consists of an outer casing, and a cotton filler 22, held in place by suitable screen means 23.

This

the tank a needle valve 24 is provided which delivers a through pipe 25 dipping into a glass vessel 26 partially filled with water to provide a seal. As a further safeguard against un-filtered air being admitted into the tank through the pipe 25, a check valve is provided at 27 which closes as soon as the discharge of compressed air from the tank ceases. A union 28 permits speedy detachment of pipe 25 and check valve 27 when the tank is being prepared for sterilization.

On the tank cover 13 is also provided a pet cock 29 which is used when the liquid or food medium 30 is bein inoculated with the mother culture. For tliis purpose the filter flask 38 shown in Fig. 6 is employed. A cotton stopper 39 filters the air drawn in while the liquid 42 is poured through the tubing 40 and pet cook 29 into the tank. A suitable stopper 41 is provided for the end of the tubing 40. For convenient testing of the contents of the tank, principally for observing the concentration of bacter1a,.a petcock 31 is provided at the bottom of the tank.

When the tank is being prepared for sterilization. the union 28 is disconnected and the checkvalve 27 and the pipe 25 removed. In its place is put the filter shown in Fi 5. This filter consists ofhalf of a union 34 into which is soldered a piece of brass tubing 33. The inside of the tubing is filled with cotton 35 and the open end is covered with cloth which may be held in place by means of a cord tied around the groove 37 in the tubing 33.

The tanks are first filled with a. certain quantity of the food medium, generally about half full. The air distributing spider Fig. 3, has previously been inserted and is centered on the bottom of the tank by a small coni cal knob and suitable recess. Next the cover 13 is put in place and screwed down air tight. The filter 21 is not attached to the valve 20 at this time. The valve 20 is then closed and the valve 24 opened. Both petcocks 29 and 31 of course are also in closed position. Then air filter 33 is attached by means of the union 28. It will be noted that now all openings are closed air tight with the exception of the needle valve 24. This valve must be left open to permit expansion and contraction of the air within the tank during the sterilization. and the cotton filter while permitting air to pass through it, keeps out all foreign organisms. The tanks are then sterilized together with the food .medium in accordance with ordinary laboratory practice.

After the sterilization the tanks are either allowed to cool or are forcefully cooled by immersing in water or otherwise. They are now ready for the inoculation. The filter flask has previously been prepared with the mother cultures or bacteria and sterilized,

and thinned to the proper consistency with clean water. Tubing 40 is connected with the petcock 29 and the mother bacteria poured Into the tank. The petcock must be now closed before the tubin is again disconnected, to avoid any unfiltered air gcttin into the tank.

he tanks are now ready for connection to the air supply. Needle valve 24 is closed, the filter 21 is attached to the globe valve 20, the small air filter 33 removed and the check valve 27 and the air seal pipe 25 put in its place.

Referring to Fig. 4, a suitable source of power A, such as an electric'motor, drives an air compressor B provided with a high pressure gauge D. From the storage tank 0 the compressed air is piped to a large cotton filter E, the air being forced through the entire length of this drum tightly filled with sterilized cotton. The air is then conducted to a reducing valve F by means of which the pressure may be cut down to a few pounds above atmospheric pressure. Another-pressure gauge G shows the low pressure. The air is then iped through manifold connections for a battery of tanks directly to the upper end of the small air-filters 21 which are attached to the globe valves 20.

After connecting a tank, the globe valve 20 is opened wide and the tank put under pressure and tested for leaks. Next the needle valve 24 is slowly opened and the flow of air through the tank is regulated principally by means of this valve.

The food medium soon becomes one great mass of bubbles which the many tiny airholes create, and the entire mass of liquid rotates slowly. Every portion of the food me dium is thus thoroughly aerated at all times and the propagation of the bacteria in this fashion is continued for about 6 days, when the desired concentration is generally reached. Progress .may be conveniently tested by drawing a few drops of the food medium through the bottom petcock 31 and placing it under the microscope.

Should the supply of compressed air be interrupted accidentally, then the check valve 27" automatically closes and the water seal 26 prevents any possibility of air entering through the needle valve 24. The water seal is also very convenient for adjusting the needle valve 24 by observing the flow in the form of bubbles.

When the desired concentration of the bacteria is reached. the air supply to the tanks in question is disconnected and the bacteria are now ready for distribution either in the liquid form as coming from the tank, or in the form of jelly which may be transported in small bottles, or prefera ly in a granular medium. By granular medium is meant a suitable rich soil of characteristics best suited for temporary homes for the bacteria.

proper proportion and the culture stored in ordinary tubs or boxes and shipped in ordinary tin. cans.

. While every conceivable precaution must be exercised to guard against contamination of-the culture during the breeding process, when the germs are few and weak, no further safeguards are required after completion of the breeding period. as the germs are then a healthy and virile state and more than able to defend themselves against forei organisms.

ithout further elaboration, the foregoing will so fully explain the gist of my invention, that others may, rent knowledge, readily adapt the same for use under various conditions of service. It will, for instance, be self-evident that the aeration of the food material may be accomplished by exhausting the air above the liquid through the valve 24 by connecting pipe 25 with a vacuum pump. -Wlien this is done, the system of piping illustrated in Figure 4 may be opened between tank G and filter E; orit may be opened between filter E, and the individual filters 21, in which case the individualfilters will be relied upon for purification of the incoming free air. These ,and many other modifications and alteragoverning the receptacle;

tions may be resorted to without eliminating certain features whichmay properly be said to constitute the essential items of new elty involved, which items are intended to be defined and secured to. me by the following. claims.

I claim: .7

1. In'combination, a' closed receptacle, a source of airunder pressure, filter means for excluding organisms or the like, from said air, connections for admittin said air to said receptacle, and flow regu ating means discharge of air from said 2. In combination, .a closed receptacle. :1. source of air under pressure communicating with said receptacle, a discharge conduitdetachably connected to said receptacle, a water seal for said discharge conduit, and a filter readily attachable 1n place of said conduit.

3. In combination, a closed receptacle, means normally keeping said rece tacle filled with air up to a redetermine pressure, connections permitting a gradual discharge to establish a flow of air through said receptacle, and 'a non return check valve in said discharge connections.

4. In combination, a closed receptacle, a source of air under pressure, means for excluding organisms from said air, means for injecting air atthe bottom of said recepthe contents thereot, and

by applying cur-' culture into means including means for injectindg --thebottom of said receptacle in the having a closed receptacle;

pressure tank receiving sterile air from said I lter, and manifold connections for delivering air to any one of a plurality of incubating units.

7. In-combination, an air com ressor, an air purifier, a storage receptacle or the pnrified air, pressure control means for maintainin a predetermined pressure in said receptac e, an incubating umt, connections for dehvermg air to saldumt to mamtam and unit" filled at a predetermined pressure, a control means for establishing a discharge predetermined rat'e of flow through said unit, and means for introducing a motherthe. contents thereof. V

8. In combination, a closed receptacle and means for rotating the contents thereof, said rection of rotation of said-contents.

9. In combination, a closed receptacle, a

source of air under pressure, means for excluding organisms from said air a connectibn for admitting said air to said receptacle and discharging the same therefrom, and means for regulating the air pressure in said receptacle to a predetermined amount,

. 10. In combination, a closed receptacle, a source of air under pressure, filter means for excluding organisms from said air-and a said unit without .contaminating 1 air at I spider supported adjacent to the bottom of I saidreceptacle and connected to said source. 11. In combination, a closed receptacle a source of air under pressure, filter means or excluding organisms from said arm, means for admitting the filtered airtosaid recep tacle, a discharge conduit leading from said receptacle, and a non-return valve 00 to said discharge conduit.

12. In combination, a closed receptacle, a

source .ofair under pressure communicating with said receptacle, a discharge conduit detachably connected to said receptacle and a non-return valve therein, and .a filter readily attachable in lace of said conduit for substitution there or for excluding organisms from said receptacle during sterilization.

In witness whereof, Ihereunto subscribe my nameithis 26 day of December, 1922. FRANK J" 

